Development Of A Cytokine Chip

细胞因子具有多种调节功能，其中最重要的功能之一是调节T细胞分化。 分泌的细胞因子谱已被证明是预测不同的T细胞分化，特别是调节细胞介导的和体液免疫的细胞因子。 为了测量不同的细胞因子的个人样本中，同时，一个基本的细胞因子芯片已经开发的基础上固相免疫亲和提取的分析物的利益。 重组链霉亲和素斑点的阵列通过羰基二咪唑桥衍生到硅烷化玻璃显微镜载玻片的表面。 抗细胞因子抗体通过其碳水化合物部分被生物素化并与链霉亲和素结合。 在分析之前用激光染料标记样品，并与芯片一起孵育。 通过洗涤除去非反应性材料后，在扫描激光密度计中读取结合的分析物。 固定化化学和保持抗体分子活性的技术的改进已经将芯片对所研究的所有50种细胞因子的灵敏度提高到1.0-2.5 pg/ml。 研究正在继续提高这种灵敏度水平，并进一步优化芯片阅读机制。 当使用细胞因子和人类临床样品的模型混合物时，芯片表现出非常好的选择性和高度的特异性。 该芯片目前正处于评估阶段，然后才能用于分析重要的调节途径，包括造血、炎症神经源性调节、细胞调节和伤口愈合。 一直在继续开发和改进用于测量临床样品中的细胞因子和生长因子的静态抗体阵列。 自动芯片阅读器的获得大大提高了抗体阵列的灵敏度和选择性。 此外，双激光系统的使用扩大了阵列的有用性。 最近与NIH临床中心重症监护中心建立的合作研究为抗体芯片开辟了新的临床应用。 目前，一组50-100种特异性抗体正在进行免疫化学评估，以纳入患者筛查芯片。 该芯片将用于筛选患有炎症性肺病的儿科患者的调节性细胞因子，疼痛相关神经肽和其他与呼吸系统疾病中免疫功能相关的调节剂。

Cytokines serve a variety of regulatory functions, one of the most important being the regulation of T-cell differentiation. Secreted cytokine profiles have been demonstrated to be predictive of different T-cell differentiation, especially cytokines regulating cell-mediated and humoral immunity. In order to measure different cytokine profiles simultaneously in individual samples, a basic cytokine chip has been developed based on solid-phase immunoaffinity extraction of the analytes of interest. Arrays of recombinant streptavidin spots were derivatized to the surfaces of silanized glass microscope slides via a carbonyldiimidazole bridge. Anti-cytokine antibodies were biotinylated via their carbohydrate moieties and bound to the steptavidin. Samples were labeled with a laser dye prior to analysis and incubated with the chip. Following removal of non-reactive materials by washing, the bound analytes were read in a scanning laser densitometer. Improvements in both the immobilization chemistry and techniques for keeping the antibody molecules active have increased the sensitivity of the chip to 1.0-2.5 pg/ml for all 50 cytokines studied. Studies are continuing to improve this level of sensitivity as well as further optimize the chip reading mechanism. The chip exhibits very good selectivity and a high degree of specificity when using both model mixtures of cytokines and human clinical samples. The chip is now in the evaluation stage prior to its use in such applications as the analysis of important regulatory pathways, including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing. Work has continued to develop and improve a static antibody array for measuring cytokines and growth factors in clinical samples. The acquisition of an automated chip reader greatly improved both sensitivity and selectivity of the antibody arrays. Additionally, the use of a dual laser system has expanded the usefulness of the array. Collaborative studies recently established with the Critical Care Center of the NIH Clinical Center have opened up new clinical applications for the antibody chip. At present, a panel of 50-100 specific antibodies is being immunochemically evaluated for inclusion in a patient-screening chip. This chip will be used to screen pediatric patients with inflammatory lung disease for regulatory cytokines, pain-associated neuropeptides, and other modulators associated with immune function in respiratory disease.